The goal of this application is to advance our understanding of the transport pathways that maintain the enterohepatic circulation of bile acids, regulate bile acid flux, and protect against bile acid-induced injury. The foundation for this proposal is our recent identification of the heteromeric Organic Solute Transporter, Osta-Ostb, as a major mechanism for intestinal bile acid transport. In this application, we will focus on the mechanisms that support Osta-Ostb to maintain bile acid homeostasis and on the cytoprotective role of Osta-Ostb in the intestinal and colonic epithelium. Our previous studies demonstrated that intestinal bile acid absorption is impaired but not abolished in Osta-/- mice. Based on those findings, the studies in Specific Aim 1 are designed to test the hypothesis that the ABC transporters Mrp3 and Mrp4 are responsible for the residual bile acid transport and serve to protect the ileal epithelium from bile acid-induced damage in the Osta null mice. Our studies demonstrated that expression of Osta-Ostb is closely regulated by substrate to balance the bile acid flux and intracellular concentration. As such, dysregulation of Osta-Ostb expression may significantly alter bile acid and lipid metabolism, underscoring the importance of the recent finding that OSTa-OSTb expression is decreased in ileum from gallstone patients. Our work showed that the regulation of bile acid synthesis is profoundly altered in Osta-/- mice. Based on these observations, the studies in Specific Aim 2 are designed to elucidate the mechanisms responsible for the altered bile acid homeostasis in the Osta null mice. In the colon, the endogenous bacterial flora convert bile acids to more hydrophobic species capable of passively diffusing into epithelial cells. However, little is known about the mechanisms engaged to export bile acids and protect the colonocyte against bile acid-induced damage. Our preliminary data suggest that Osta-/- mice are more susceptible to dextran sodium sulfate-induced colonic injury as compared to littermate controls. These findings underscore the importance of the recent observation that OSTa expression is dramatically reduced in colon from patients with ulcerative colitis. Based on these findings, the studies in Specific Aim 3 are designed to test the hypothesis that Osta-Ostb is responsible for colonic basolateral bile acid transport and functions to protect the colon from the cytotoxic effects of bile acids. The long-term goal of this work is to understand the mechanisms that control the compartmentalization and enterohepatic circulation of bile acids and their relationship to human gastrointestinal and metabolic disease.